Histone protein H2A and variant H2A.X play central roles in the cellular response to DNA double-strand breaks (DSBs) in mammalian chromatin. Near DSBs, chromatin becomes enriched in H2A.X which gets phosphorylated by ATM kinase. Phosphorylation of H2A.X sets off a cascade of post-translational modifications (PTMs) culminating with the recruitment of RING-finger E3 ubiquitin ligase RNF168 to DNA damage sites. RNF168 and cognate E2 ubiquitin-conjugating enzyme UbcH5c catalyze the mono- ubiquitylation of histones H2A and H2A.X at lysine residues 13 and 15 (H2AK13ub and H2AK15ub) in the nucleosome core particle. These two PTMs are recognized by and determine the recruitment to damaged chromatin of several DNA damage response (DDR) proteins including 53BP1, BRCA1/RAP80, RAD18 and RNF169 that control the balance between the DSB repair pathways of non-homologous end joining (NHEJ) and homologous recombination (HR). The goal of this research program is to determine the molecular mechanisms by which H2A and H2A.X are involved in the DDR. Using NMR spectroscopy, X-ray crystallography, cryo-electron microscopy, biochemistry and cell biology, we will probe how RNF168-UbcH5c generates H2AK13ub and H2AK15ub and how these PTMs are recognized by HR-promoting DDR proteins in the context of the nucleosome. We will also test the hypothesis that ubiquitylation of H2A.X at lysine residues 13 or 15 directly contributes to chromatin decompaction, thereby facilitating the recruitment of DNA repair proteins to DSBs. Finally, we will address the mechanism of H2A/H2A.X exchange and H2A.X enrichment at DSBs promoted by human histone chaperone FACT. Collectively, our work will provide fundamental knowledge relevant to NHEJ and HR repair mechanisms with important implications for human health as explained in the Narrative paragraph. As is often the case in research, it is likely that new unanticipated and fascinating questions will arise during our studies that, in keeping with the MIRA R35 mechanism, we will tackle as needed.